Uses of synthetic peptides corresponding to telopeptide sequences of cross-linked type l collagen metabolites

ABSTRACT

Methods of determining collagen degradation in vivo, by quantitating the concentration of a peptide in a body fluid, the peptide being a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link or a type III collagen telopeptide containing a hydroxylysyl pyridinoline cross-link. Suitable methods include immunometric assays, fluorometric assays, and electrochemical titrations for quantitation. The structures of specific peptides having cross-links and kits for quantitating these peptides in a body fluid are described.

[0001] This is a continuation-in-part of U.S. Ser. No. 444,881, filedDec. 1, 1989, which is a continuation-in-part of U.S. Ser. No. 118,234,filed Nov. 6, 1987.

[0002] This invention was made with government support under grantsAR37318 and AR36794 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to methods for detecting andmonitoring collagen degradation in vivo. More specifically, it relatesto methods for quantitating cross-linked telopeptides produced in vivoupon degradation of collagen types II and III.

BACKGROUND OF THE INVENTION

[0004] Three known classes of collagens have been described to date. TheClass I collagens, subdivided into types I, II, III, V, and XI, areknown to form fibrils. These collagens are all synthesized asprocollagen molecules, made up of N-terminal and C-terminal propeptides,which are attached to the core collagen molecule. After removal of thepropeptides, which occurs naturally in vivo during collagen synthesis,the remaining core of the collagen molecule consists largely of atriple-helical domain having terminal telopeptide sequences which arenontriple-helical. These telopeptide sequences have an importantfunction as sites of intermolecular cross-linking of collagen fibrilsextracellularly.

[0005] The present invention relates to methods of detecting collagendegradation based on assaying for particular cross-linked telopeptidesproduced in vivo upon collagen degradation. In the past, assays havebeen developed for monitoring degradation of collagen in vivo bymeasuring various biochemical markers, some of which have beendegradation products of collagen. For example, bone turnover associatedwith Paget's disease has been monitored by measuring small peptidescontaining hydroxyproline, which are excreted in the urine followingdegradation of bone collagen. Russell et al., Metab. Bone Dis. and Rel.Res. 4 and 5, 255-262 (1981); and Singer, F. R., et al., Metabolic BoneDisease, Vol. II (eds. Avioli, L. V. and Kane, S. M.), 489-575 (1978),Academic Press, New York.

[0006] Other researchers have measured the cross-linking compoundpyridinoline in urine as an index of collagen degradation in jointdisease. See, for background and for example, Wu and Eyre, Biochemistry,23:1850 (1984); Black et al., Annals of the Rheumatic Diseases,48:641-644 (1989); Robins et al.; Annals of the Rheumatic Diseases,45:969-973 (1986); and Seibel et al., The Journal of Rheumatology,16:964 (1989). In contrast to the present invention, some priorresearchers have hydrolyzed peptides from body fluids and then lookedfor the presence of individual hydroxypyridinium residues. None of theseresearchers has reported measuring a telopeptide containing a cross-linkthat is naturally produced in vivo upon collagen degradation, as in thepresent invention.

[0007] U.K. Patent application GB 2,205,643 reports that the degradationof type III collagen in the body is quantitatively determined bymeasuring the concentration of an N-terminal telopeptide from type IIIcollagen in a body fluid. In this reference, it is reported thatcross-linked telopeptide regions are not desirable. In fact, thisreference reports that it is necessary to use a non-cross-linked sourceof collagen to obtain the telopeptide. The peptides of the presentinvention are all cross-linked. Collagen cross-links are discussed ingreater detail below, under the heading “Collagen Cross-Linking.”

[0008] There are a number of reports indicating that collagendegradation can be measured by quantitating certain procollagenpeptides. The present invention involves telopeptides rather thanpropeptides, the two being distinguished by their location in thecollagen molecule and the timing of their cleavage in vivo. See U.S.Pat. No. 4,504,587; U.S. Pat. No. 4,312,853; Pierard et al., AnalyticalBiochemistry 141:127-136 (1984); Niemela, Clin. Chem., 31/8:1301-1304(1985); and Rohde et al., European Journal of Clinical Investigation,9:451-459 (1979).

[0009] U.S. Pat. No. 4,778,768 relates to a method of determiningchanges occurring in articular cartilage involving quantifyingproteoglycan monomer or antigenic fragments thereof in a synovial fluidsample. This patent does not relate to detecting cross-linkedtelopeptides derived from degraded collagen.

[0010] Dodge, J. Clin. Invest., 83:647-661 (1981) discloses methods foranalyzing type II collagen degradation utilizing a polyclonal antiserumthat specifically reacts with unwound alpha-chains and cyanagenbromide-derived peptides of human and bovine type II collagens. Thepeptides involved are not cross-linked telopeptides as in the presentinvention.

[0011] Amino acid sequences of human type III collagen, human proα1(II)collagen, and the entire preproα1(III) chain of human type III collagenand corresponding cDNA clones have been investigated and determined byseveral groups of researchers. See Loidl et al., Nucleic Acids Research12:9383-9394 (1984); Sangiorgi et al., Nucleic Acids Research,13:2207-2225 (1985); Baldwin et al., Biochem. J., 262:521-528 (1989);and Ala-Kokko et al., Biochem. J., 260:509-516 (1989). None of thesereferences specifies the structures of particular telopeptidedegradation products that could be measured to determine the amount ofdegraded fibrillar collagen in vivo.

[0012] In spite of the above-described background information, thereremains a need for effective and simple assays for determining collagendegradation in vivo. Such assays could be used to detect and monitordisease states in humans, such as osteoarthritis (type II collagendegradation), and various inflammatory disorders, such as vasculitissyndrome (type III collagen degradation).

[0013] Assays for type I collagen degradation, described in the parentapplication, U.S. Ser. No. 118,234, can be utilized to detect and assessbone resorption in vivo. Detection of bone resorption may be a factor ofinterest in monitoring and detecting diseases such as osteoporosis.Osteoporosis is the most common bone disease in man. Primaryosteoporosis, with increased susceptibility to fractures, results from aprogressive net loss of skeletal bone mass. It is estimated to affect15-20 million individuals in the United States. Its basis is anage-dependent imbalance in bone remodeling, i.e., in the rates ofsynthesis and degradation of bone tissue.

[0014] About 1.2 million osteoporosis-related fractures occur in theelderly each year including about 538,000 compression fractures of thespine, about 227,000 hip fractures and a substantial number of earlyfractured peripheral bones. Twelve to 20% of the hip fractures are fatalbecause they cause severe trauma and bleeding, and half of the survivingpatients require nursing home care. Total costs fromosteoporosis-related injuries now amount to at least $7 billion annually(Barnes, O. M., Science, 236:914 (1987)).

[0015] Osteoporosis is most common in postmenopausal women who, onaverage, lose 15% of their bone mass in the 10 years after menopause.This disease also occurs in men as they get older and in youngamenorrheic women athletes. Despite the major, and growing, social andeconomic consequences of osteoporosis, no method is available formeasuring bone resorption rates in patients or normal subjects. A majordifficulty in monitoring the disease is the lack of a specific assay formeasuring bone resorption rates.

[0016] Methods for assessing bone mass often rely on measuringwhole-body calcium by neutron activation analysis or mineral mass in agiven bone by photon absorption techniques. These measurements can giveonly long-term impressions of whether bone mass is decreasing. Measuringcalcium balances by comparing intake with output is tedious, unreliableand can only indirectly appraise whether bone mineral is being lost overthe long term. Other methods currently available for assessing decreasedbone mass and altered bone metabolism include quantitative scanningradiometry at selected bone locations (wrist, calcaneus, etc.) andhistomorphometry of iliac crest biopsies. The former provides a crudemeasure of the bone mineral content at a specific site in a single bone.Histomorphometry gives a semi-quantitative assessment of the balancebetween newly deposited bone seams and resorbing surfaces.

[0017] A urinary assay for the whole-body output of degraded bone in 24hours would be much more useful. Mineral studies (e.g., calcium balance)cannot do this reliably or easily. Since bone resorption involvesdegradation of the mineral and the organic matrix, a specificbiochemical marker for newly degraded bone products in body fluids wouldbe the ideal index. Several potential organic indices have been tested.For example, hydroxyproline, an amino acid largely restricted tocollagen, and the principal structural protein in bone and all otherconnective tissues, is excreted in urine. Its excretion rate is known tobe increased in certain conditions, notably Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased, as pointedout above. For this reason, urinary hydroxyproline has been usedextensively as an amino acid marker for collagen degradation. Singer, F.R., et al. (1978), cited hereinabove.

[0018] U.S. Pat. No. 3,600,132 discloses a process for determination ofhydroxyproline in body fluids such as serum, urine, lumbar fluid andother intercellular fluids in order to monitor deviations in collagenmetabolism. In particular, this inventor notes that in pathologicconditions such as Paget's disease, Marfan's syndrome, osteogenesisimperfecta, neoplastic growth in collagen tissues and in various formsof dwarfism, increased collagen anabolism or catabolism as measured byhydroxyproline content in biological fluids can be determined. Thisinventor measures hydroxyproline by oxidizing it to a pyrrole compoundwith hydrogen peroxide and N-chloro-p-toluenesulphonamide followed bycolorimetric determination in p-dimethyl-amino-benzaldehyde.

[0019] In the case of Paget's disease, the increased urinaryhydroxyproline probably comes largely from bone degradation;hydroxyproline, however, generally cannot be used as a specific index.Much of the hydroxyproline in urine may come from new collagen synthesis(considerable amounts of the newly made protein are degraded andexcreted without ever becoming incorporated into tissue fabric), andfrom turnover of certain blood proteins as well as other proteins thatcontain hydroxyproline. Furthermore, about 80% of the freehydroxyproline derived from protein degradation is metabolized in theliver and never appears in the urine. Kiviriko, K. I. Int. Rev. Connect.Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin.Invest. 48: 1 (1969).

[0020] Hydroxylysine and its glycoside derivatives, both peculiar tocollagenous proteins, have been considered to be more accurate thanhydroxyproline as markers of collagen degradation. However, for the samereasons described above for hydroxyproline, hydroxylysine and itsglycosides are probably equally non-specific markers of bone resorption.Krane, S. M. and Simon, L. S. Develop. Biochem., 22:185 (1981).

[0021] In addition to amino acids unique to collagen, variousnon-collagenous proteins of bone matrix such as osteocalcin, or theirbreakdown products, have formed the basis of immunoassays aimed atmeasuring bone metabolism. Price, P. A. et al. J. Clin. Invest., 66: 878(1980), and Gundberg, C. M. et al., Meth. Enzymol., 107:516 (1984).However, it is now clear that bone-derived non-collagenous proteins,though potentially a useful index of bone metabolic activity areunlikely, on their own, to provide quantitative measures of boneresorption. The concentration in serum of osteocalcin, for example,fluctuates quite widely both normally and in metabolic bone disease. Itsconcentration is elevated in states of high skeletal turnover but it isunclear whether this results from increased synthesis or degradation ofbone. Krane, S. M., et al., Develop. Biochem., 22:185 (1981), Price, P.A. et al., J. Clin. Invest., 66:878 (1980); and Gundberg, C. M. et al.,Meth. Enzymol., 107:516 (1984).

[0022] Collagen Cross-Linking

[0023] The polymers of most genetic types of vertebrate collagen requirethe formation of aldehyde-mediated cross-links for normal function.Collagen aldehydes are derived from a few specific lysine orhydroxylysine side-chains by the action of lysyl oxidase. Various di-,tri- and tetrafunctional cross-linking amino acids are formed by thespontaneous intra- and intermolecular reactions of these aldehydeswithin the newly formed collagen polymers; the type of cross-linkingresidue varies specifically with tissue type (see Eyre, D. R. et al.,Ann. Rev. Biochem., 53:717-748 (1984)).

[0024] Two basic pathways of cross-linking can be differentiated for thebanded (67 nm repeat) fibrillar collagens, one based on lysinealdehydes, the other on hydroxylysine aldehydes. The lysine aldehydepathway dominates in adult skin, cornea, sclera, and rat tail tendon andalso frequently occurs in other soft connective tissues. Thehydroxylysine aldehyde pathway dominates in bone, cartilage, ligament,most tendons and most internal connective tissues of the body, Eyre, D.R. et al. (1974) vida supra. The operating pathway is governed bywhether lysine residues are hydroxylated in the telopeptide sites wherealdehyde residues will later be formed by lysyl oxidase (Barnes, M. J.et al., Biochem. J., 139:461 (1974)).

[0025] The chemical structure(s) of the mature cross-linking amino acidson the lysine aldehyde pathway are unknown, but hydroxypyridiniumresidues have been identified as mature products on the hydroxylysinealdehyde route. On both pathways and in most tissues the intermediate,borohydride-reducible cross-linking residues disappear as the newlyformed collagen matures, suggesting that they are relatively short-livedintermediates (Bailey, A. J. et al., FEBS Lett., 16:86 (1971)).Exceptions are bone and dentin, where the reducible residues persist inappreciable concentration throughout life, in part apparently becausethe rapid mineralization of the newly made collagen fibrils inhibitsfurther spontaneous cross-linking interactions (Eyre, D. R., In: TheChemistry and Biology of Mineralized Connective Tissues, (Veis, A. ed.)pp. 51-55 (1981), Elsevier, New York, and Walters, C. et al., Calc.Tiss. Intl., 35:401-405 (1983)).

[0026] Two chemical forms of 3-hydroxypyridinium cross-link have beenidentified (Formula I and II). Both compounds are naturally fluorescent,with the same characteristic excitation and emission spectra (Fujimoto,D. et al. Biochem. Biophys. Res. Commun., 76:1124 (1977), and Eyre, D.R., Develop. Biochem., 22:50 1981)). These amino acids can be resolvedand assayed directly in tissue hydrolysates with good sensitivity usingreverse phase HPLC and fluorescence detection. Eyre, D. R. et al.,Analyte. Biochem., 137:380-388 (1984). It should be noted that thepresent invention involves quantitating particular peptides rather thanamino acids.

[0027] In growing animals, it has been reported that these maturecross-links may be concentrated more in an unmineralized fraction ofbone collagen than in the mineralized collagen (Banes, A. J., et al.,Biochem. Biophys. Res. Commun., 113:1975 (1983). However, other studieson young bovine or adult human bone do not support this concept, Eyre,D. R., In: The Chemistry and Biology of Mineralized Tissues (Butler, W.T. ed.) p. 105 (1985), Ebsco Media Inc., Birmingham, Ala.

[0028] The presence of collagen hydroxypyridinium cross-links in humanurine was first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. andBoucek, R. J., Biochem J., 197:759-762 (1981)) using lengthy isolationprocedures for peptides and conventional amino acid analysis. At thattime, they were aware only of the HP form of the cross-link. Robins(Robins, S. P., Biochem J., 207:617-620 (1982) has reported anenzyme-linked immunoassay to measure HP in urine, having raisedpolyclonal antibodies to the free amino acid conjugated to bovine serumalbumin. This assay is intended to provide an index for monitoringincreased joint destruction that occurs with arthritic diseases and isbased, according to Robins, on the finding that pyridinoline is muchmore prevalent in cartilage than in bone collagen.

[0029] In more recent work involving enzyme-linked immunoassay, Robinsreports that lysyl pyridinoline is unreactive toward antiserum topyridinoline covalently linked to bovine serum albumin (Robins et al.,Ann. Rheum. Diseases, 45:969-973 (1986)). Robins' urinary index forcartilage destruction is based on the discovery that hydroxylysylpyridinoline, derived primarily from cartilage, is found in urine atconcentrations proportional to the rate of joint cartilage resorption(i.e., degradation). In principle, this index could be used to measurewhole body cartilage loss; however, no information on bone resorptionwould be available.

[0030] A need therefore exists for a method that allows the measurementof whole-body bone resorption rates in humans. The most useful suchmethod would be one that could be applied to body fluids, especiallyurine. The method should be sensitive, i.e., quantifiable down to 1picomole and rapidly measure 24-hour bone resorption rates so that theprogress of various therapies (e.g., estrogen) can be assessed.

SUMMARY OF THE INVENTION

[0031] The present invention is based on the discovery of the presenceof particular cross-linked telopeptides in body fluids of patients andnormal human subjects. These telopeptides are produced in vivo duringcollagen degradation and remodeling. The term “telopeptides” is used ina broad sense herein to mean cross-linked peptides having sequences thatare associated with the telopeptide region of, e.g., type II and typeIII collagens and which may have cross-linked to them a residue orpeptide associated with the collagen triple-helical domain. Generally,the telopeptides disclosed herein will have fewer amino acid residuesthan the entire telopeptide domains of type II and type III collagens.Typically, the telopeptides of the present invention will comprise twopeptides linked by a pyridinium cross-link and further linked by apyridinium cross-link to a residue or peptide of the collagentriple-helical domain. Having disclosed the structures of thesetelopeptides herein, it will be appreciated by one of ordinary skill inthe art that they may also be produced other than in vivo, e.g.,synthetically. These peptides will generally be provided in purifiedform, e.g., substantially free of impurities, particularly otherpeptides.

[0032] The present invention also relates to methods for determining invivo degradation of type II and type III collagens. The methods involvequantitating in a fluid the concentration of particular telopeptidesthat have a 3-hydroxypyridinium cross-link and that are derived fromcollagen degradation. The methods disclosed in the present invention areanalogous to those previously disclosed in U.S. Ser. No. 118,234, filedNov. 6, 1987, for determining the absolute rate of bone resorption invivo. Those methods involved quantitating in a body fluid theconcentration of telopeptides having a 3-hydroxypyridinium cross-linkderived from bone collagen resorption.

[0033] In a representative assay, the patient's body fluid is contactedwith an immunological binding partner specific to a telopeptide having a3-hydroxypyridinium cross-link derived from type II or type IIIcollagen. The body fluid may be used as is or purified prior to thecontacting step. This purification step may be accomplished using anumber of standard procedures, including cartridge adsorption andelution, molecular sieve chromatography, dialysis, ion exchange, aluminachromatography, hydroxyapatite chromatography, and combinations thereof.

[0034] Other representative embodiments of quantitating theconcentration of peptide fragments having a 3-hydroxypyridiniumcross-link in a body fluid include electrochemical titration, naturalfluorescence spectroscopy, and ultraviolet absorbance. Electrochemicaltitration may be conducted directly upon a body fluid without furtherpurification. However, when this is not possible due to excessivequantities of contaminating substances, the body fluid is first purifiedprior to the electrochemical titration step. Suitable methods forpurification prior to electrochemical detection include dialysis, ionexchange chromatography, alumina chromatography, molecular sievechromatography, hydroxyapatite chromatography and ion exchangeabsorption and elution.

[0035] Fluorometric measurement of a body fluid containing a3-hydroxypyridinium cross-link is an alternative way of quantitatingcollagen degradation (and, hence, bone resorption, if type I peptidesare quantitated). The fluorometric assay can be conducted directly on abody fluid without further purification. However, for certain bodyfluids, particularly urine, it is preferred that purification of thebody fluid be conducted prior to the fluorometric assay. Thispurification step consists of dialyzing an aliquot of a body fluid suchas urine against an aqueous solution thereby producing partiallypurified peptide fragments retained within the nondiffusate (retentate).The nondiffusate is then lyophilized, dissolved in an ion pairingsolution and adsorbed onto an affinity chromatography column. Thechromatography column is washed with a volume of ion pairing solutionand, thereafter, the peptide fragments are eluted from the column withan eluting solution. These purified peptide fragments may then behydrolyzed and the hydrolysate resolved chromatographically.Chromatographic resolution may be conducted by either high-performanceliquid chromatography or microbore high performance liquidchromatography.

[0036] The invention includes peptides having structures identical topeptides derived from collagen degradation, substantially free fromother human peptides, which may be obtained from a body fluid. Thepeptides contain at least one 3-hydroxypyridinium cross-link, inparticular, a lysyl pyridinoline cross-link or a hydroxylysylpyridinoline cross-link, and are derived from the telopeptide region oftype II or type III collagen linked to one or more residues from atriple-helical domain, typically by the action of endogenous proteasesand/or peptidases.

[0037] The structures of the type II and type III telopeptides aredisclosed below. Information on the type I telopeptides, originallypresented in U.S. Ser. No. 118,234, is also included.

[0038] Another aspect of the present invention involves assays for thepeptides described herein in which the pyridinium rings are intact andcleaved. Since it is suspected that some cleavage of pyridinium ringsoccurs in vivo, assays that detect both intact and cleaved pyridiniumrings may lead to more accurate assessments of collagen degradation. Inconnection with this aspect of the present invention, specific bindingpartners to the individual peptides containing intact or cleavedpyridinium rings, may be employed in the assays. Individual specificbinding partners that recognize both types of peptides (both intact andcleaved pyridinium ring containing peptides) may be employed.Alternatively, specific binding partners that discriminate betweenpeptides containing the intact pyridinium ring and those in which thepyridinium ring is cleaved, could also be used.

[0039] Structure of Cross-Linked Telopeptides Derived from Type ICollagen

[0040] A specific telopeptide having a 3-hydroxypyridinium cross-linkderived from the N-terminal (amino-terminal) telopeptide domain of bonetype I collagen has the following amino acid sequence:

[0041] is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln isglutamine or pyrrolidine carboxylic acid.

[0042] The invention also encompasses a peptide containing at least one3-hydroxypyridinium cross-link derived from the C-terminal(carboxy-terminal) telopeptide domain of bone type I collagen. TheseC-terminal telopeptide sequences are cross-linked with either lysylpyridinoline or hydroxylysyl pyridinoline. An example of such a peptidesequence is represented by the formula:

[0043] is hydroxylysyl or lysyl pyridinoline.

[0044] Since the filing of U.S. Ser. No. 118,234, the inventor hasdiscovered evidence of two additional type I collagen telopeptides inbody fluids, having the following structures:

[0045] These telopeptides may also be quantitated in body fluids inaccordance with the present invention.

[0046] Structure of a Cross-Linked Telopeptide Derived from Type IICollagen

[0047] A specific telopeptide having a hydroxylysyl pyridinolinecross-link derived from the N-terminal telopeptide domain of type IICollagen has the following amino acid sequence (referred to hereinbelowas the core peptide structure):

[0048] wherein the cross-linking residue depicted as Hyl-Hyl-Hyl ishydroxylysyl pyridinoline (HP), a natural 3-hydroxypyridinium residuepresent in mature collagen fibrils of various tissues.

[0049] Carboxy-terminal peptides from type II collagen have not beendetected in body fluids, and it is suspected that potential peptidesderived from the C-terminal telopeptide region of type II collagen aresubstantially degraded in vivo, perhaps all the way to the free HPcross-linking amino acid.

[0050] Structure of Cross-Linked Telopeptides Derived from Type IIICollagen

[0051] By analogy to the above disclosure, cross-linked peptides thatare derived from proteolysis of human type III collagen may be presentin body fluids. These peptides have a core structure embodied in thefollowing parent structures:

[0052] is hydroxylysyl or lysyl pyridinoline, and Gln is glutamine orpyrrolidine carboxylic acid.

[0053] A likely cross-linked peptide derived from type III collagen inbody fluids has the core structure:

[0054] that is derived from two α1(III)N-telopeptide domains linked toan hydroxylysyl pyridinoline residue (Hyl-Hyl-Hyl).

[0055] A second possible fragment of the C-telopeptide cross-linkingdomain, based on the collagen types I and II peptides observed in urine,has the core structure:

[0056] Smaller and larger versions (differing by one to three aminoacids on each component chain) of these two peptides corresponding tothe parent sequences shown above (FORMULAE VIII and IX) may also bepresent and measurable in body fluids. Analogous smaller and largerversions of each of the peptides disclosed herein form part of thepresent invention as well.

[0057] The invention generally includes all specific binding partners tothe peptides described herein. “Specific binding partners” are moleculesthat are capable of binding to the peptides of the present invention.Included within this term are immunological binding partners, such asantibodies (monoclonal and polyclonal), antigen-binding fragments ofantibodies (e.g., Fab and F(ab′)₂ fragments), single-chainantigen-binding molecules, and the like, whether made by hybridoma orrDNA technologies.

[0058] The invention includes fused cell hybrids (hybridomas) thatproduce monoclonal antibodies specific for the above-described collagenpeptides having 3-hydroxypyridinium cross-links (both with an intactpyridinium ring and one that has been cleaved).

[0059] The invention further includes monoclonal antibodies produced bythe fused cell hybrids, and those antibodies (as well as bindingfragments thereof, e.g., Fab) coupled to a detectable marker. Examplesof detectable markers include enzymes, chromophores, fluorophores,coenzymes, enzyme inhibitors, chemiluminescent materials, paramagneticmetals, spin labels, and radioisotopes. Such specific binding partnersmay alternatively be coupled to one member of a ligand-binding partnercomplex (e.g., avidin-biotin), in which case the detectable marker canbe supplied bound to the complementary member of the complex.

[0060] The invention also includes test kits useful for quantitating theamount of peptides having 3-hydroxypyridinium cross-links derived fromcollagen degradation in a body fluid. The kits may include a specificbinding partner to a peptide derived from degraded collagen as disclosedherein. The specific binding partners of the test kits may be coupled toa detectable marker or a member of a ligand-binding partner complex, asdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061]FIG. 1 is a depiction of type II collagen and a proposal for thesource of telopeptides. It is not established whether the twotelopeptides shown come from one collagen molecule as depicted in FIG. 1or from two collagen molecules.

[0062]FIG. 2 shows relative fluorescence (297 nm excitation; 390 nmemission) versus fraction number (4 ml), obtained during molecular sievechromatographic purification of cross-linked telopeptides. Cross-linkedtype II collagen telopeptides are contained in the fractions designatedII.

[0063]FIG. 3A shows relative fluorescence (330 nm excitation, 390 nmemission) versus elution time of fractions during ion exchange HPLC(DEAE-5PW). Cross-linked type II collagen telopeptides are contained inthe fraction designated IV.

[0064]FIG. 3B shows absorbance (220 nm) versus elution time in minutesfor the same chromatogram.

[0065]FIG. 4A shows relative fluorescence (297 nm excitation, 390 nmemission) versus elution time of fractions during reverse phase HPLC.Cross-linked type II collagen telopeptides are eluted as indicated. Thefractions indicated by the bar (—) show evidence by sequence andcomposition analysis of the peptides indicated that retain or have lostthe gly (G) and pro (P) residues.

[0066]FIG. 4B shows absorbance (220 nm) as a function of elution timeduring reverse phase HPLC.

[0067]FIG. 5 compares the concentration of HP and LP in both corticaland cancellous human bone with age.

[0068]FIG. 6 depicts the cross-link molar ratios of HP to LP as afunction of age.

[0069]FIG. 7A shows relative fluorescence (297 nm excitation, >370 nmemission) as a function of elution volume during reverse phase HPLCseparation of cross-linked type I collagen N-telopeptides.

[0070]FIG. 7B shows relative fluorescence (297 nm excitation, >370 nmemission) versus elution volume during reverse phase HPLC separation ofcross-linked type I collagen C-telopeptides.

[0071]FIG. 8A shows relative fluorescence (297 nm excitation, >380 nmemission) as a function of elution time for the cross-linked type Icollagen telopeptides.

[0072]FIG. 8B shows relative fluorescence (297 nm excitation, >380 nmemission) as a function of elution time for the cross-linked type Icollagen telopeptides.

[0073]FIG. 9 shows results of binding experiments with therepresentative monoclonal antibody HB ______ and: the P1 peptide(Formula III herein, open squares); an α2 (I) N-telopeptide (QYDGKGVGC,solid diamonds); and an α1 (I) N-telopeptide (YDEKSTGGC, solid squares).

[0074]FIG. 10 shows a portion of the structure of the N-telopeptideregion of decalcified human bone collagen. The P1 peptide (Formula III)is enclosed in a box; it contains an epitope that correlates with boneresorption.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0075] Type II Collagen Telopeptides

[0076] The core peptide structure of the type II collagen peptides maybe found in body fluids as a component of larger peptides that bearadditional amino acids or amino acid sequences on one or more ends ofthe three peptide sequences joined by the HP residue. FIG. 1 shows howtype II collagen telopeptides, which are linked to a triple-helicalsequence, may be produced in vivo from a human source using theproteolytic enzymes pepsin and trypsin. Smaller fragments that have lostamino acids from the core peptide structure, particularly from thehelical sequence, may also occur in body fluids. Generally, additions ordeletions of amino acids from the core peptide structure will involvefrom 1 to about 3 amino acids. Additional amino acids will generally bedetermined by the type II collagen telopeptide sequence that occursnaturally in vivo. As examples, peptides having the following structure:

[0077] can be isolated chromatographically from urine, and another ofstructure:

[0078] may also be isolated. In addition, glycosylated variants of thecore structure and its larger and smaller variants may occur in which agalactose residue or a glucosyl galactose residue are attached to theside chain hydroxyl group of the HP cross-linking residue. Each peak inthe graph shown in FIGS. 4A and 4B may correspond to a cross-linkedfragment of particular structure that may be quantitated for purposes ofthe present invention.

[0079] These structures are consistent with their site of origin inhuman type II collagen fibrils at a molecular cross-linking site formedbetween two α1(II) C-telopeptides and residue 87 of a triple-helicaldomain, the known sequences about which are:

[0080] The isolated peptide fragments represent the products ofproteolytic degradation of type II collagen fibrils within the body. Thecore structure containing the HP residue is relatively resistant tofurther proteolysis and provides a quantitative measure of the amount oftype II collagen degraded.

[0081] Collagen type II is present in hyaline cartilage of joints in theadult skeleton. Quantitation of the collagen type II telopeptides in abody fluid, for example by way of a monoclonal antibody that recognizesan epitope in the peptide structure, would provide a quantitativemeasure of whole-body cartilage destruction or remodeling. In apreferred embodiment, the present invention involves an assay forcartilage tissue degradation in humans based on quantifying the urinaryexcretion rate of at least one member of this family of telopeptides.Such an assay could be used, for example, to:

[0082] (1) screen adult human subjects for those individuals havingabnormally high rates of cartilage destruction as an early diagnosticindicator of osteoarthritis;

[0083] (2) monitor the effects of potential antiarthritic drugs oncartilage metabolism in osteoarthritic and rheumatoid arthriticpatients; or

[0084] (3) monitor the progress of degenerative joint disease inpatients with osteoarthritis and rheumatoid arthritis and theirresponses to various therapeutic interventions.

[0085] Osteoarthritis is a degenerative disease of the articulatingcartilages of joints. In its early stages it is largely non-inflammatory(i.e. distinct from rheumatoid arthritis). It is not a single diseasebut represents the later stages of joint failure that may result fromvarious factors (e.g. genetic predisposition, mechanical overusage,joint malformation or a prior injury, etc.). Destruction of jointarticular cartilage is the central progressive feature ofosteoarthritis. The incidence of osteoarthritis, based on radiographicsurveys, ranges from 4% in the 18-24 year age group to 85% in the 75-79year age group. At present the disease can only be diagnosed by pain andradiographic or other imaging signs of advanced cartilage erosion.

[0086] The assays disclosed above may be used to detect early evidenceof accelerated cartilage degradation in mildly symptomatic patients, tomonitor disease progress in more advanced patients, and as a means ofmonitoring the effects of drugs or other therapies.

[0087] In normal young adults (with mature skeletons) there is probablyvery little degradation of cartilage collagen. A test that could measurefragments of cartilage collagen in the urine (and in the blood and jointfluid) would be very useful for judging the “health” of cartilage in thewhole body and in individual joints. The type II collagen-specificpeptide assays described above will accomplish this. In the long term,such an assay could become a routine diagnostic screen for spottingthose individuals whose joints are wearing away. They could be targetedearly on for preventative therapy, for example, by the next generationof so-called chondroprotective drugs now being evaluated by the majorpharmaceutical companies who are all actively seeking better agents totreat osteoarthritis.

[0088] Other diseases in which joint cartilage is destroyed include:rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, Reiter's syndrome, relapsingpolychondritis, the low back pain syndrome, and other infectious formsof arthritis. The type II collagen-specific assays described hereincould be used to diagnose and monitor these diseases and evaluate theirresponse to therapy, as disclosed above in connection withosteoarthritis.

[0089] Type III Collagen Telopeptides

[0090] As pointed out above, human type III collagen telopeptides thatmay be present in body fluids are expected to have a core structureembodied in the following parent structures:

[0091] is hydroxylysyl pyridinoline.

[0092] By analogy to the type II peptides, the type III collagenpeptides may occur in glycosylated forms of the core structure. Forexample, galactose residues or glucosylgalactose residues may beattached to the core structure, e.g. by way of hydroxyl groups.

[0093] The cross-linking residue of the type III collagen peptides isdepicted as a 3-hydroxypyridinium residue, hydroxylysyl pyridinoline.The type II telopeptide structures have been found to primarily havehydroxylysyl pyridinoline cross-linking residues. However, whereas thetype II collagen peptides are derived from the N-terminal telopeptideregion of type II collagen, the type III collagen peptides may bederived from either the N-terminal or the C-terminal of type IIIcollagen, as long as at least one cross-linking residue is present.

[0094] Type III collagen is present in many connective tissues inassociation with type I collagen. It is especially concentrated invascular walls, in the skin and in, for example, the synovial membranesof joints where its accelerated turnover might be observed ininflammatory joint diseases such as rheumatoid arthritis.

[0095] A specific assay for type III collagen degradation byquantitating cross-linked type III collagen peptides as disclosed above,can be used for detecting, diagnosing, and monitoring variousinflammatory disorders, possibly with particular application to thevasculitis syndromes. In conjunction with assays for measuring bone typeI and cartilage type II collagen degradation rates, such an assay couldbe used as a differential diagnostic tool for patients with variousdegenerative and inflammatory disorders that result in connective tissuedestruction or pathological processes.

[0096] Isolation of Type II and Type III Collagen Telopeptides

[0097] General Procedure:

[0098] Urine is collected form a normal adolescent during a rapid phaseof skeletal growth. Using a sequence of chromatographic steps thatinclude but are not limited to, adsorption on selective cartridges of ahydrophobic interaction support and an ion-exchange support andmolecular sieve, ion-exchange and reverse-phase HPLC columnchromatography steps, individual peptides are isolated. The cross-linkedpeptides containing HP (and LP) residues are detected during columnchromatography by their natural fluorescence (Ex max 297 nm<pH 4, Ex max330 nm, >pH 6; Em max 390 nm). An exemplary isolation procedure isprovided in the Example below.

SPECIFIC EXAMPLE:

[0099] Fresh urine (at 4° C.) diluted 5 times with water and adjusted to2% (v/v) trifluoroacetic acid, passed through a C-18 hydrophobic bindingcartridge (Waters C-18 Sep-pak prewetted with 80% (v/v) acetonitrilethen washed with water). Retained peptides were washed with water theneluted with 3 ml of 20% (v/v) acetonitrile, and this eluent was adjustedto 0.05 M NH₄HCO₃, 10% (v/v) acetonitrile by addition of an equal volumeof 0.1 M NH₄HCO₃. This solution was passed through a QMA-Sep-pak(Waters), which was washed with 10 ml of 0.1M NaCl, 20% (v/v)acetonitrile followed by 10 ml of water and the peptides were theneluted with 3 ml of 1% (v/v) trifluoroacetic acid and dried by Speed-Vac(Savant).

[0100] Peptides were fractionated in three chromatographic steps. Thefirst step was molecular sieve chromatography on a column of Bio-GelP-10 (Bio Rad Labs, 2.5 cm×90 cm) eluted by 10% (v/v) acetic acid,monitoring the effluent for HP fluorescence as shown in FIG. 2. In FIG.2, the Y-axis is the relative fluorescence emission at 390 nm (297 nmexcitation), and the X-axis is the fraction number. The fraction sizewas 4 ml. The fractions indicated as II are enriched in the cross-linkedcollagen type II telopeptides. The cross-linked collagen type Itelopeptides are contained in the fractions indicated as III and IV.Fractions spanning pool II (enriched in the type II collagencross-linked peptides) were combined, freeze-dried and fractionated byion-exchange column chromatography on a DEAE-HPLC column (TSK-DEAE-5PW,7.5 mm×7.5 mm, Bio-Rad Labs), equilibrated with 0.02 M Tris/HCl, 10%(v/v) acetonitrile, pH 7.5 and eluted with a gradient of 0-0.5M NaCl inthe same buffer as shown in FIG. 2.

[0101]FIG. 3A plots relative fluorescence emission at 390 nm (330 nmexcitation) versus elution time. The cross-linked collagen type IItelopeptides are found primarily in the segment indicated as IV. FIG. 3Bplots absorbance at 220 nm as a function of elution time in minutes.Pool IV contains the type II collagen cross-linked peptides. Individualpeptides were then resolved from pool IV by reverse phase HPLC on a C-18column (Aquapore RP-300, 25 cm×4.6 mm, Brownlee Labs), eluting with agradient of 0-30% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid.FIG. 4A shows a plot of relative fluorescence intensity at 390 nm (297nm excitation) as a function of elution time. The peaks associated withparticular peptides are indicated in FIG. 4A. FIG. 4B shows the relativeabsorbance at 220 nm as a function of time.

[0102] Cross-linked peptide fragments of type III collagen containing HPcross-linking residues may be isolated by a similar combination of stepsfrom the urine of normal growing subjects or, for example, from theurine of patients with inflammatory disorders of the vasculature.

[0103] Type I Collagen Telopeptides

[0104] This aspect of the invention is based on the discovery that bothlysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) peptidefragments (i.e., telopeptides, as used herein) derived from reabsorbedbone collagen are excreted in the urine without being metabolized. Theinvention is also based on the discovery that no other connectivetissues contain significant levels of LP and that the ratio of HP to LPin mature bone collagen remains relatively constant over a person'slifetime.

[0105]FIG. 5 compares the concentration of HP and LP in both corticaland cancellous human bone with age. It is observed that theconcentration of HP plus LP cross-links in bone collagen reaches amaximum by age 10 to 15 years and remains reasonably constant throughoutadult life. Furthermore, the ratio of HP to LP, shown in FIG. 6, showslittle change throughout life, remaining constant at about 3.5 to 1.These baseline data demonstrate that the 3-hydroxypyridinium cross-linksin bone collagen remains relatively constant and therefore that bodyfluids derived from bone collagen degradation will contain3-hydroxypyridinium cross-linked peptide fragments at concentrationsproportional to the absolute rate of bone resorption.

[0106] Since LP is the 3-hydroxypyridinium cross-link unique to bonecollagen, the method for determining the absolute rate of boneresorption, in its simplest form, is based on quantitating theconcentration of peptide fragments containing 3-hydroxypyridiniumcross-links and preferably lysyl pyridinoline (LP) cross-links in a bodyfluid.

[0107] As used in this description and in the appended claims withrespect to type I, II, or III telopeptides, by “quantitating” is meantmeasuring by any suitable means, including but not limited tospectrophotometric, gravimetric, volumetric, coulometric, immunometric,potentiometric, or amperometric means the concentration of peptidefragments containing 3-hydroxypyridinium cross-links in an aliquot of abody fluid. Suitable body fluids include urine, serum, and synovialfluid. The preferred body fluid is urine.

[0108] Since the concentration of urinary peptides will decrease as thevolume of urine increases, it is further preferred that when urine isthe body fluid selected, the aliquot assayed be from a combined pool ofurine collected over a fixed period of time, for example, 24 hours. Inthis way, the absolute rate of bone resorption or collagen degradationis calculated for a 24 hour period. Alternatively, urinary peptides maybe measured as a ratio relative to a marker substance found in urinesuch as creatinine. In this way the urinary index of collagendegradation and bone resorption would remain independent of urinevolume.

[0109] In one embodiment of the present invention, monoclonal orpolyclonal anti-bodies are produced which are specific to the peptidefragments containing lysyl pyridinoline cross-links found in a bodyfluid such as urine. Type I telopeptide fragments may be isolated from abody fluid of any patient, however, it is preferred that these peptidesare isolated from patients with Paget's disease or from rapidly growingadolescents, due to their high concentration of type I peptidefragments. Type II and type III telopeptides may be isolated from a bodyfluid of any patient but may be more easily obtained from patientssuffering from diseases involving type II or type III collagendegradation or from rapidly growing adolescents.

[0110] Isolation of Type I Collagen Telopeptides

[0111] Urine from patients with active Paget's disease is dialyzed inreduced porosity dialysis tubing (<3,500 mol. wt. cut off Spectropore)at 4° C. for 48 h to remove bulk solutes. Under these conditions thepeptides of interest are largely retained. The freeze-driednon-diffusate is then eluted (200 mg aliquots) from a column (90 cm×2.5cm) of Bio-Gel P2 (200-400 mesh) in 10% acetic acid at room temperature.A region of effluent that combines the cross-linked peptides is definedby measuring the fluorescence of collected fractions at 297 nmexcitation/395 nm emission, and this pool is freeze-dried. Furtherresolution of this material is obtained on a column of Bio-Gel P-4(200-400 mesh, 90 cm×2.5 cm) eluted in 10% acetic acid.

[0112] Two contiguous fraction pools are defined by monitoring thefluorescence of the eluant above. The earlier fraction is enriched inpeptide fragments having two amino acid sequences that derive from theC-terminal telopeptide domain of the αI(I) chain of bone type I collagenlinked to a third sequence derived from the triple-helical body of bonetype I collagen. These three peptide sequences are cross-linked with3-hydroxypyridinium. The overlapping later fraction is enriched inpeptide fragments having an amino acid sequence that is derived from theN-terminal telopeptide domain of bone type I collagen linked through a3-hydroxypyridinium cross-links.

[0113] Individual peptides are then resolved from each of the twofractions obtained above by ion-exchange HPLC on a TSK DEAE-5-PW column(Bio Rad 7.5 cm×7.5 mm) eluting with a gradient of NaCl (0-0.2M) in0.02M Tris-HCl, pH 7.5 containing 10% (v/v) acetonitrile. The N-terminaltelopeptide-based and C-terminal telopeptide-based cross-linked peptideselute in a series of 3-4 peaks of fluorescence between 0.08M and 0.15MNaCl. The C-terminal telopeptide-based cross-linked peptides elute firstas a series of fluorescent peaks, and the major and minor N-terminaltelopeptide-based cross-linked peptides elute towards the end of thegradient as characteristic peaks. Each of these is collected,freeze-dried and chromatographed on a C-18 reverse phase HPLC column(Vydac 218TP54, 25 cm×4.6 mm) eluted with a gradient (0-10%) ofacetonitrile: n-propanol (3:1 v/v) in 0.01M trifluoroacetic acid. About100-500 μg of individual peptide fragments containing3-hydroxypyridinium cross-links can be isolated by this procedure from asingle 24 h collection of Paget's urine.

[0114] Amino acid compositions of the major isolated peptides confirmedpurity and molecular sizes by the whole number stoichiometry ofrecovered amino acids. N-terminal sequence analysis by Edman degradationconfirmed the basic core structures corresponding to the sequences ofthe known cross-linking sites in type I collagen and from the matchingamino acid compositions. The N-terminal telopeptide sequence of theα2(I) chain was blocked from sequencing analysis due presumably to theknown cyclization of the N-terminal glutamine to pyrrolidone carboxylicacid.

[0115] A typical elution profile of N-terminal telopeptides obtained bythe above procedure is shown in FIG. 7A. The major peptide fragmentobtained has an amino acid composition:(Asx)₂(Glx)₂(Gly)₅Val-Tyr-Ser-Thr, where Asx is the amino acid Asp orAsn and Glx is the amino acid Gln or Glu. The sequence of this peptideis represented by Formula III below.

[0116] The C-terminal telopeptide-based cross-linked peptides resolvedby reverse phase HPLC as described above are shown in FIG. 7B. As can beseen from this figure, these peptides are further resolved into a seriesof C-terminal telopeptides each containing the 3-hydroxypyridiniumcross-links. The major peptide, shown in FIG. 7B, was analyzed asdescribed above and was found to have the amino acid composition:(Asp)₅(Glu)₄(Gly)₁₀(His)₂(Arg)₂(Hyp)₂(Ala)₅. The sequence of thispeptide is represented by formula IV below. It is believed that theother C-terminal telopeptide-based cross-linked peptides appearing asminor peaks in FIG. 7B represent additions and deletions of amino acidsto the structure shown in Formula IV. Any of the peptides containedwithin these minor peaks are suitable for use as immunogens as describedbelow.

[0117] represents the HP or LP cross-links and Gln represents glutamineor pyrrolidone carboxylic acid.

[0118] Equivalents of the peptides represented by the above structures,in terms of their presence in a body fluid due to collagen degradation,include those cases where there is some variation in the peptidestructure. Examples of such variation include 1-3 amino acid additionsto the N and C termini as well as 1-3 terminal amino acid deletions. Forexample, a peptide corresponding to Formula III, but having a tyrosineresidue attached to the amino terminus of the N-terminal aspartateresidue has been detected in relatively minor quantities in human urine.Smaller peptide fragments of the molecule represented by Formula IVderived from bone resorption are especially evident in urine. These arefound in the minor peaks of the C-terminal telopeptide fraction seen inFIG. 7B and can be identified by amino acid composition and sequenceanalysis.

EXAMPLES OF PROCEDURES FOR QUANTITATING PEPTIDES

[0119] A. Immunological Procedure for Quantitating Peptides

[0120] Immunological binding partners capable of specifically binding topeptide fragments derived from bone collagen obtained from aphysiological fluid can be prepared by methods well known in the art.The preferred method for isolating these peptide fragments is describedabove. By immunological binding partners as used herein is meantantibodies and antibody fragments capable of binding to a telopeptide.

[0121] Both monoclonal and polyclonal antibodies specifically bindingthe peptides disclosed herein and their equivalents are prepared bymethods known in the art. For example, Campbell, A. M. LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 13 (1986).Elsevier, herein incorporated by reference. It is possible to produceantibodies to the above peptides or their equivalents as isolated.However, because the molecular weights of these peptide fragments aregenerally less than 5,000, it is preferred that the hapten be conjugatedto a carrier molecule. Suitable carrier molecules include, but are notlimited to, bovine serum albumin, ovalbumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). Preferred carriers are thyroglobulin and KLH.

[0122] It is well known in the art that the orientation of the hapten,as it is bound to the carrier protein, is of critical importance to thespecificity of the anti-serum. Furthermore, not all hapten-proteinconjugates are equally successful immunogens. The selection of aprotocol for binding the particular hapten to the carrier proteintherefore depends on the amino acid sequence of the urinary peptidefragments selected. For example, if the peptide represented by FormulaIII is selected, a preferred protocol involves coupling this hapten tokeyhole limpet hemocyanin (KLH), or other suitable carrier, withglutaraldehyde. An alternative protocol is to couple the peptides to KLHwith a carbodiimide. These protocols help to ensure that the preferredepitope (discussed below under the heading “Characteristics of aPreferred Epitope)” are presented to the primed vertebrate antibodyproducing cells (e.g., B lymphocytes).

[0123] Other peptides, depending on the source, may require differentbinding protocols. Accordingly, a number of binding agents may besuitably employed. These include, but are not limited to, carbodiimides,glutaraldehyde, mixed anhydrides, as well as both homobifunctional andheterobifunctional reagents (see for example the Pierce 1986-87 catalog,Pierce Chemical Co., Rockford, Ill.). Preferred binding agents includecarbodiimides and heterobifunctional reagents such asm-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).

[0124] Methods for binding the hapten to, the carrier molecule are knownin the art. See for example, Chard, T., Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 6 (1987) Partz Elsevier, N.Y.,herein incorporated by reference.

[0125] Either monoclonal or polyclonal antibodies to the hapten-carriermolecule immunogen can be produced. However, it is preferred thatmonoclonal antibodies (MAb) be prepared. For this reason it is preferredthat immunization be carried out in the mouse. Immunization protocolsfor the mouse usually include an adjuvant. Examples of suitableprotocols are described by Chard, T. (1987) vida supra. Spleen cellsfrom the immunized mouse are harvested and homogenized and thereafterfused with cancer cells in the presence of polyethylene glycol toproduce a fused cell hybrid which produces monoclonal antibodiesspecific to peptide fragments derived from collagen. Examples of suchpeptides are represented by the formulas given above. Suitable cancercells include myeloma, hepatoma, carcinoma, and sarcoma cells. Detaileddescriptions of this procedure, including screening protocols, protocolsfor growing selected hybrid cells and harvesting monoclonal antibodiesproduced by the selected hybrid cells are provided in Galfre, G. andMilstein, C., Meth. Enzymol., 73:1 (1981). A preferred preliminaryscreening protocol involves the use of peptide fragments derived frombone collagen resorption and containing 3-hydroxypyridinium cross-linksin a solid phase radioimmunoassay. A specific example describing apreferred monoclonal antibody is provided below.

[0126] The monoclonal antibodies or other immunological binding partnersused in connection with the present are preferably specific for aparticular type of collagen telopeptide. For example, assays for thetype II or type III collagen degradation telopeptides should preferablybe able to distinguish between the type I, type II, and type IIIpeptides. However, in some cases, such selectivity will not benecessary, for example, if it is known that a patient is not sufferingdegradation of one type of collagen but is suspected of sufferingdegradation from the assayed type of collagen. Because of thedifferences in amino acid sequences between the type I, type II, andtype III families of telopeptides, cross-reactivity should not occur toa significant degree. Indeed, hybridomas can be selected for during thescreening of splenocyte fusion clones that produce monoclonal antibodiesspecific for the cross-linked telopeptide of interest (and lack affinityfor those of the other two collagen types). Based on the differences insequence of the isolated peptide structures, such specificity isentirely feasible. Peptide fragments of the parent types I, II and IIIcollagens, suitable for such hybridoma screening, can be prepared fromhuman bone, cartilage and other tissues and used to screen clones frommice immunized appropriately with the individual cross-linked peptideantigens isolated from body fluid.

[0127] Immunological binding partners, especially monoclonal antibodies,produced by the above procedures, or equivalent procedures, are employedin various immunometric assays to quantitate the concentration of thepeptides having 3-hydroxypyridinium cross-links described above. Theseimmunometric assays preferably comprise a monoclonal antibody orantibody fragment coupled to a detectable marker. Examples of suitabledetectable markers include but are not limited to: enzymes, coenzymes,enzyme inhibitors, chromophores, fluorophores, chemiluminescentmaterials, paramagnetic metals, spin labels, and radionuclides. Examplesof standard immunometric methods suitable for quantitating thetelopeptides include, but are not limited to, enzyme linkedimmunosorbent assay (ELISA) (Ingvall, E., Meth. Enzymol., 70 (1981)),radio-immunoassay (RIA), and “sandwich” immunoradiometric assay (IRMA).

[0128] In its simplest form, these immunometric methods can be used todetermine the absolute rate of bone resorption or collagen degradationby simply contacting a body fluid with the immunological binding partnerspecific to a collagen telopeptide having a 3-hydroxypyridiniumcross-link.

[0129] It is preferred that the immunometric assays described above beconducted directly on untreated body fluids (e.g. urine, blood, serum,or synovial fluid). Occasionally, however, contaminating substances mayinterfere with the assay necessitating partial purification of the bodyfluid. Partial purification procedures include, but are not limited to,cartridge adsorption and elution, molecular sieve chromatography,dialysis, ion exchange, alumina chromatography, hydroxyapatitechromatography and combinations thereof.

[0130] Test kits, suitable for use in accordance with the presentinvention, contain specific binding partners such as monoclonalantibodies prepared as described above, that specifically bind topeptide fragments derived from collagen degradation found in a bodyfluid. It is preferred that the specific binding partners of this testkit be coupled to a detectable marker of the type described above. Testkits containing a panel of two or more specific binding partners,particularly immunological binding partners, are also contemplated. Eachimmunological binding partner in such a test kit will preferably notcross-react substantially with a telopeptide, derived from another typeof collagen. For example, an immunological binding partner that bindsspecifically with a type II collagen telopeptide should preferably notcross-react with either a type I or type III collagen telopeptide. Asmall degree (e.g., 5-10%) of cross-reactivity may be tolerable. Othertest kits may contain a first specific binding partner to acollagen-derived telopeptide having a cross-link containing a pyridiniumring (which may be OH-substituted), and a second specific bindingpartner to a telopeptide having the same structure as the firsttelopeptide except that the pyridinium ring has been cleaved, such asphotolytically.

[0131] (i) Monoclonal Antibody Production

[0132] The following is an example of preparation of a monoclonalantibody against a peptide immunogen based on Formula III above.

[0133] A fraction enriched in the peptide of Formula III (indicative ofbone collagen degradation) was prepared from adolescent human urineusing reverse phase and molecular sieve chromatography. The peptide wasconjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde usingstandard procedures. Mice (Balb/c) were immunized subcutaneously withthis conjugate (50-70 μg), first in complete Freund's adjuvant, thenboosted (25 μg) at 3 weekly intervals in incomplete Freund's adjuvantintraperitoneally. After test bleeds had shown a high titer against theFormula III peptide (referred to herein as P1) conjugated to bovineserum albumin (BSA) using an ELISA format, selected mice were boostedwith a low dose (5 μg) of the immunogen in sterile PBS intravenously.Three days later, cells from the spleens of individual mice were fusedwith mouse myeloma cells using standard hybridoma technology. Thesupernatants of hybridoma clones growing in individual wells of 96-wellplates were screened for reactive monoclonal antibodies, initially usinga crude P1 preparation conjugated to BSA. After formal cloning bylimiting dilution, the antibodies produced by individual hybridomas werecharacterized against a panel of screening antigens using ELISAanalysis. These antigens were the P1 (Formula III) and P2 (Formula VII)peptides conjugated to BSA. An inhibition assay was used in which P1conjugated to BSA was plated out in the plastic wells, and antibody waspre-incubated with a solution of the potential antigen. A secondaryantibody (goat anti-mouse IgG conjugated to horseradish peroxidase, HRP)was used for color development using an appropriate substrate. Adesirable monoclonal antibody with high binding affinity for the P1peptide was identified. When used as an ascites fluid preparation, theantibody worked in an inhibition assay with optimal color yield at 2million-fold dilution (which indicates a binding constant in the rangeof 10⁻⁹ to 10⁻¹¹ M⁻¹, most likely about 10⁻¹⁰ M⁻¹). In an ELISA format,the antibody was able to detect and measure P1 present in normal humanurine without any concentration or clean-up steps. The hybridoma thatproduces this preferred monoclonal antibody has been deposited at theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, under accession number HB ______. This hybridomais designated below as 1H11; the monoclonal antibody it produces isdesignated below as MAb-1H11.

[0134] Sandwich assays were also shown to work using the P1-specificmonoclonal antibody and a polyclonal antiserum raised in rabbits againstconjugated P1. Either P1-specific monoclonal antibodies, polyclonalantiserum, binding fragments thereof, or the like can be used to bindspecifically to P1 from urine, in a detectable manner using standardELISA and other immunoassay protocols.

[0135] (ii) Characteristics of a Preferred Epitope

[0136] The epitope recognized by the antibody MAb-1H11 is embodied inthe structure of P1. The epitope is recognized in pure P1 and in certainlarger peptides that contained the P1 structure (e.g., P1 attached to atyrosine residue via the N-terminal aspartate residue of P1). Theepitope includes chemical features of both of the two telopeptidesequences embodied in the structure of peptide P1. Peptides synthesizedto match the human α1 (I) and α2 (I) N-telopeptide sequences, with theaddition of a C-terminal cysteine for coupling to bovine serum albumin(i.e., YDEKSTGGC and QYDGKGVGC), were not recognized by MAb-1H11. Thiswas shown by ELISA using the free peptides competing against plated-outP1 (see FIG. 9) or directly as binding partners conjugated to BSA andplated out. Referring to FIG. 9, the absorbance at λ=450 nm of adetectable marker is plotted against the concentration of free P1peptide. As the amount of free P1 increases, the amount of detectablemarker bound to immobilized (plated-out) P1 diminishes. In comparison,the α2(I) and α1(I) N-telopeptides demonstrate little if any significantcompetitive binding with MAb-1H11.

[0137] In addition, a larger form of P1 bearing a tyrosine residue onthe N-terminal aspartic acid was recovered from urine by affinitybinding to MAb-1H11, but in lower yield than P1. Other slightly largerpeptides bearing the P1 epitope were also recovered but in even smalleramounts.

[0138] The antibody was not selective for the nature of the cross-linkin P1, i.e., whether hydroxylysyl pyridinoline (HP) or lysylpyridinoline (LP). Both HP-containing and LP-containing forms werebound, apparently with equal affinity, judging by the analysis ofpeptides isolated from urine by an affinity column consisting ofMAb-1H11 coupled to agarose.

[0139] The free cross-linking amino acids, HP and LP, either made byacid hydrolysis from bone collagen or as present naturally in urine werenot recognized by MAb-1H11. After photolytic opening of the 3-pyridinolring in peptide P1 with UV light (long UV wavelengths), specificantibody binding was also unaffected, presumably because the individualpeptides remained cross-linked to each other. The epitope recognized byMAb-1H11, therefore, is made up of at least a combination of chemicaland conformational features embodied in the two telopeptide sequencesshown boxed in FIG. 10, together with steric features imposed by thetrivalent cross-linking amino acid that links them. The α2 (I) Ntelopeptide sequence, QYDGK, is a particularly significant part of theepitope.

[0140] The fact that the epitope recognized by MAb-1H11 does not dependon an intact pyridinium ring is an unexpected discovery. If ring-openingoccurs either in vivo or even in vitro under routine handlingconditions, as appears likely, then a quantitative assay of the subjectpeptide(s) having intact pyridinium rings will underestimate the amountof bone resorption. Preliminary observations indicate that degradationof pyridinium rings in the subject peptides appears to occurparticularly in urine and/or in urine samples, even if refrigerated.Accordingly, an assay based on the present disclosure is expected to becomparatively more accurate. Two embodiments are envisioned: a singlespecific binding partner is employed that recognizes both closed andopen-ringed embodiments of the targeted peptide(s); or two specificbinding partners are employed, which differentiate between the closed-and open-ringed epitopes, respectively.

[0141] Further experiments showed that the epitope resides in human bonecollagen but is exposed and bound by MAb-1H11 only after extensiveproteolysis. Thus, peptides produced from decalcified human bonecollagen by bacterial collagenase were bound by MAb-1H11 and shown to bederived from the N-telopeptide to helix site shown in FIG. 10. One formcontained the hexapeptide GIKGHR (in place of the non-telopeptide K armin P1), which is clearly derived from α1 (I) residues 928-933. Anotherform embodied an equivalent but distinct hexapeptide that was derivedfrom the α2 (I) chain. Fragments of human bone collagen solubilized bypepsin, CNBr, or trypsin were not recognized by MAb-1H11, either in anELISA format when used as competitive inhibitors or on a Western blotafter SDA-polyacrylamide electrophoresis, indicating that thesesolubilizing agents do not produce the epitope recognized by MAb-1H11.

[0142] B. Electrochemical Procedure for Assaying for Peptides

[0143] An alternative procedure for assaying for the above-describedpeptides consists of measuring a physical property of the peptideshaving 3-hydroxypyridinium cross-links. One such physical propertyrelies upon electrochemical detection. This method consists of injectingan aliquot of a body fluid, such as urine, into an electrochemicaldetector poised at a redox potential suitable for detection of peptidescontaining the 3-hydroxypyridinium ring. The 3-hydroxypyridinium ring,being a phenol, is subject to reversible oxidation and therefore theelectrochemical detector (e.g., Model 5100A Coulochem sold by Esa 45Wiggins Ave., Bedford, Mass.) is a highly desirable instrument suitablefor quantitating the concentration of the present peptides. Two basicforms of electrochemical detector are currently commercially available:amperometric (e.g., BioAnalytical Systems) and coulometric (ESA, Inc.,Bedford, Mass. 01730). Both are suitable for use in accordance with thepresent invention, however, the latter system is inherently moresensitive and therefore preferred since complete oxidation or reductionof the analyzed molecule in the column effluent is achieved. Inaddition, screening or guard electrodes can be placed “upstream” fromthe analytical electrode to selectively oxidize or reduce interferingsubstances thereby greatly improving selectivity. Essentially, thevoltage of the analytical electrode is tuned to the redox potential ofthe sample molecule, and one or more pretreatment cells are set todestroy interferents in the sample.

[0144] In a preferred assay method, a standard current/voltage curve isestablished for standard peptides containing lysyl pyridinoline orhydroxylysyl pyridinoline in order to determine the proper voltage toset for optimal sensitivity. This voltage is then modified dependingupon the body fluid, to minimize interference from contaminants andoptimize sensitivity. Electrochemical detectors, and the optimumconditions for their use are known to those skilled in the art. Complexmixtures of body fluids can often be directly analyzed with theelectrochemical detector without interference. Accordingly, for mostpatients no pretreatment of the body fluid is necessary. In some caseshowever, interfering compounds may reduce the reliability of themeasurements. In such cases, pretreatment of the body fluid (e.g.,urine) may be necessary.

[0145] Accordingly, in an alternative embodiment of the invention, abody fluid is first purified prior to electrochemically titrating thepurified peptide fragments. The purification step, may be conducted in avariety of ways including but not limited to dialysis, ion exchangechromatography, alumina chromatography, hydroxyapatite chromatography,molecular sieve chromatography, or combinations thereof. In a preferredpurification protocol, a measured aliquot (25 ml) of a 24 hour urinesample is dialyzed in reduced porosity dialysis tubing to remove thebulk of contaminating fluorescent solutes. The non-diffusate is thenlyophilized, redissolved in 1% heptafluorobutyric acid (HFBA), an ionpairing solution, and the peptides adsorbed on a Waters Sep-Pak C-18cartridge. This cartridge is then washed with 5 ml of 1% HFBA, and theneluted with 3 ml of 50% methanol in 1% HFBA.

[0146] Another preferred method of purification consists of adsorbing ameasured aliquot of urine onto an ion-exchange adsorption filter andeluting the adsorption filter with a buffered eluting solution. Theeluate fractions containing peptide fragments having 3-hydroxypyridiniumcross-links are then collected to be assayed.

[0147] Still another preferred method of purification employs molecularsieve chromatography. For example, an aliquot of urine is applied to aBio-Gel P2 or Sephadex G-20 column and the fraction eluting in the1000-5000 Dalton range is collected. It will be obvious to those-skilledin the art that a combination of the above methods may be used to purifyor partially purify urine or other body fluids in order to isolate thepeptide fragments having 3-hydroxypyridinium cross-links. The purifiedor partially purified peptide fragments obtained by the above proceduresmay be subjected to additional purification procedures, furtherprocessed or assayed directly in the partially purified state.Additional purification procedures include resolving partially purifiedpeptide fragments employing high performance liquid chromatography(HPLC) or microbore HPLC when increased sensitivity is desired. Thesepeptides may then be quantitated by electrochemical titration.

[0148] A preferred electrochemical titration protocol consists of tuningthe redox potential of the detecting cell of the electrochemicaldetector (Coulochem Model 5100A) for maximum signal with pure HP. Thedetector is then used to monitor the effluent from a C-18 HPLC columnused to resolve the partially purified peptides.

[0149] C. Fluorometric Procedure for Quantitating Peptides

[0150] An alternative preferred method for quantitating theconcentration of peptides having 3-hydroxypyridinium cross-links asdescribed herein is to measure the characteristic natural fluorescenceof these peptides. For those body fluids containing few naturallyoccurring fluorescent materials other than the 3-hydroxypyridiniumcross-links, fluorometric assay may be conducted directly withoutfurther purification of the body fluid. In this case, the peptides areresolved by HPLC and the natural fluorescence of the HP and LP aminoacid residues is measured at 395 nm upon excitation at 297 nm,essentially as described by Eyre, D. R., et al., Analyte. Biochem.137:380 (1984), herein incorporated by reference.

[0151] It is preferred, in accordance with the present invention, thatthe fluorometric assay be conducted on urine. Urine, however, usuallycontains substantial amounts of naturally occurring fluorescentcontaminants that must be removed prior to conducting the fluorometricassay. Accordingly, urine samples are first partially purified asdescribed above for electrochemical detection. This partially purifiedurine sample can then be fluorometrically assayed as described above.Alternatively, the HP and LP cross-linked peptides in the partiallypurified urine samples or other body fluids can be hydrolyzed in 6M HClat about 108° C. for approximately 24 hours as described by Eyre, et al.(1984) vida supra. This process hydrolyzes the amino acids connected tothe lysine precursors of “tripeptide” HP and LP cross-links, producingthe free HP and LP amino acids represented by Formulae I and II. Thesesmall “tripeptides” are then resolved by the techniques described above,preferably by HPLC, and the natural fluorescence is measured (Ex 297 nm,Ex 390 nm).

[0152] Optionally, the body fluid (preferably urine) is passed directlythrough a C-18 reverse phase affinity cartridge after addingacetonitrile/methanol 5 to 10% V/V. The non-retentate is adjusted to0.05-0.10M with a cationic ion-pairing agent such as tetrabutyl ammoniumhydroxide and passed through a second C-18 reverse phase cartridge. Thewashed retentate, containing fluorescent peptides, from this secondcartridge is eluted with acetonitrile:water (or methanol:water), driedand fluorescent peptides are analyzed by reverse phase HPLC or microboreHPLC using an anionic ion-pairing agent such as 0.01M trifluoroaceticacid in the eluant.

[0153]FIG. 8A displays the elution profile resolved by reverse phaseHPLC of natural fluorescence for a hydrolysate of peptide fragments fromnormal human urine. Measurement of the integrated area within theenvelope of a given component is used to determine the concentration ofthat component within the sample. The ratio of HP:LP found in normalhuman urine and urine from patients having Paget's disease, FIG. 8B, areboth approximately 4.5:1. This is slightly higher than the 4:1 ratiofound in bone itself (Eyre, et al., 1984). The higher ratio found inurine indicates that a portion of the HP fraction in urine may come fromsources other than bone, such as the diet, or other sources of collagendegradation, i.e., cartilage catabolism. It is for this reason that itis preferred that LP which derives only from bone be used to provide anabsolute index of bone resorption. However, in the absence of excessivecartilage degradation such as in rheumatoid arthritis or in cases wherebone is rapidly being absorbed, HP or a combination of HP plus LP may beused as an index of bone resorption.

[0154] While the invention has been described in conjunction withpreferred embodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by theappended claims and equivalents thereof.

1 6 1 9 PRT Artificial Sequence PEPTIDE (1)..(9) corresponds to sequenceof cross-linked type I collagen metabolite 1 Gln Tyr Asp Gly Lys Gly ValGly Cys 1 5 2 9 PRT Artificial Sequence PEPTIDE (1)..(9) corresponds tosequence of cross-linked type I collagen metabolite 2 Tyr Asp Glu LysSer Thr Gly Gly Cys 1 5 3 5 PRT Artificial Sequence PEPTIDE (1)..(5)corresponds to sequence of cross-linked type I collagen metabolite 3 GlnTyr Asp Gly Lys 1 5 4 6 PRT Artificial Sequence PEPTIDE (1)..(6)corresponds to sequence of cross-linked type I collagen metabolite 4 GlyIle Lys Gly His Arg 1 5 5 7 PRT Artificial Sequence PEPTIDE (1)..(7)corresponds to sequence of cross-linked type I collagen metabolite 5 AspGlu Lys Ser Thr Gly Gly 1 5 6 8 PRT Artificial Sequence PEPTIDE (1)..(8)corresponds to sequence of cross-linked type I collagen metabolite 6 GlnTyr Asp Gly Lys Gly Val Gly 1 5

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of determiningbone resorption, comprising the step of contacting a body fluid with atleast one specific binding partner to a first peptide consistingessentially of the structure

is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamineor pyrrolidine carboxylic acid, and to a second peptide having the aboveformula except that the pyridinium ring of the cross-linking amino acidhas been cleaved.
 2. A method according to claim 1, wherein said bodyfluid is urine, blood, serum, or synovial fluid.
 3. A method accordingto claim 1, comprising contacting said body fluid with a first specificbinding partner to said first peptide and a second specific bindingpartner to said second peptide.
 4. A method according to claim 3,wherein said first and second specific binding partners are monoclonalantibodies.
 5. A method according to claim 1, comprising contacting saidbody fluid with one specific binding partner that is capable of bindingspecifically to each of said peptides.
 6. A method according to claim 5,wherein said specific binding partner is a monoclonal antibody.
 7. A kitfor determining bone resorption, comprising a specific binding partnerto a first peptide consisting essentially of the structure

is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamineor pyrrolidine carboxylic acid, and to a second peptide having the aboveformula except that the pyridinium ring of the cross-linking amino acidhas been cleaved.
 8. A kit according to claim 7, wherein said specificbinding partners are monoclonal antibodies.
 9. A method of determiningcartilage degradation in vivo, comprising quantitating in a body fluidthe concentrations of a first peptide comprising a C-terminal type IIcollagen telopeptide containing a hydroxylysyl pyridinoline cross-linkwhich has an intact pyridinium ring, and a second peptide comprising aC-terminal type II collagen telopeptide containing a hydroxylysylpyridinoline cross-link which has a cleaved pyridinium ring.
 10. Amethod of determining cartilage degradation according to claim 9,wherein the body fluid is urine, blood, serum, or synovial fluid.
 11. Amethod of determining cartilage degradation according to claim 9,wherein the detecting step comprises contacting the body fluid with afirst specific binding partner to said first peptide and a secondspecific binding partner to said second peptide.
 12. A method ofdetermining cartilage degradation according to claim 9, wherein thefirst and second peptides have the structure:

is hydroxylysyl pyridinoline, said first and second peptides containingan intact or a cleaved pyridinium ring, respectively.
 13. A method ofdetermining cartilage degradation according to claim 9, wherein thefirst and second peptides have the structure:

is hydroxylysyl pyridinoline, said first and second peptides containingan intact or a cleaved pyridinium ring, respectively.
 14. A method ofdetermining collagenous connective tissue degradation in vivo,comprising quantitating in a body fluid the concentrations of a firstpeptide comprising a type III collagen telopeptide containing a3-hydroxypyridinium cross-link, and a second peptide comprising a typeIII collagen telopeptide containing a 3-hydroxypyridinium cross-linkwhich has a cleaved pyridinium ring.
 15. The method according to claim14, wherein the 3-hydroxypyridinium cross-link is hydroxylysylpyridinoline.
 16. The method according to claim 14, wherein the bodyfluid is urine, blood, serum, or synovial fluid.
 17. The methodaccording to claim 14, wherein the detecting step comprises contactingthe body fluid with a first specific binding partner to said firstpeptide and a second specific binding partner to said second peptide.18. The method according to claim 7, wherein the type III collagentelopeptide is:

is hydroxylysyl pyridinoline, said first and second peptides containingan intact or a cleaved pyridinium ring, respectively.
 19. A kit forcarrying out the method of any of claims 9-18, comprising at least twospecific binding partners to said first and second peptides.
 20. A cellline that produces a specific binding partner that binds to first andsecond peptides consisting essentially of the structure

is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamineor pyrrolidine carboxylic acid, and wherein the pyridinium ring of thecross-linking amino acid in the first and second peptides is closed andopen, respectively.
 21. The cell line of claim 20, having theidentifying characteristics of ATCC HB ______ (1H11).
 22. The specificbinding partner produced by the cell line of claim
 20. 23. A monoclonalantibody produced by the cell line of claim 21.